Quantitative PCR Analysis of Gene Expression

Quantitative polymerase chain reaction (qPCR) was performed as previously described (Liu et al., 2021 (link); Liu et al., 2022 (link)). Briefly, total RNA was isolated using TRIzol reagent (Invitrogen, United States) and concentration was measured using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, United States). Then RNA was reversely transcribed into cDNA using Color Reverse Transcription Kit (EZBioscience, United States), and qPCR was performed on Bio-Rad CFX-96 (Bio-Rad, United States) with Color SYBR Green qPCR Master Mix (EZBioscience, United States). The 2^^-△CT value was calculated and the distal 2^^-△CT value was divided by the proximal 2^^-△CT value, and the results were normalized. The qPCR primers used in the study are listed in Supplementary Table S1.

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Liu X., Shao Y., Han L., Zhu Y., Tu J., Ma J., Zhang R., Yang Z, & Chen J. (2024). Microbiota affects mitochondria and immune cell infiltrations via alternative polyadenylation during postnatal heart development. Frontiers in Cell and Developmental Biology, 11, 1310409.

Publication 2023

TRIzol reagent NanoDrop 2000 Color Reverse Transcription Kit Bio-Rad CFX-96 Color SYBR Green qPCR Master Mix

Corresponding Organization : Sun Yat-sen University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

2^-ΔCT value
Normalized results (distal 2^-ΔCT divided by proximal 2^-ΔCT)

control variables

Total RNA isolation using TRIzol reagent
RNA concentration measurement using NanoDrop 2000 spectrophotometer
Reverse transcription of RNA into cDNA using Color Reverse Transcription Kit
QPCR performed on Bio-Rad CFX-96 with Color SYBR Green qPCR Master Mix

controls

None explicitly mentioned

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